Cripto blocking antibodies and uses thereof

ABSTRACT

The invention provides Cripto blocking antibodies, or biologically functional fragments thereof, and uses thereof. Antibodies which bind Cripto and modulate Cripto signaling are provided. Antibodies which bind Cripto and block the interaction between Cripto and ALK4 are provided. Antibodies which bind Cripto and modulate tumor growth are also provided. Antibodies which bind Cripto, modulate signaling, and modulate tumor growth are also provided. Antibodies which bind Cripto, block the interaction between Cripto and ALK4 and modulate tumor growth are provided. The invention also provides methods of using these antibodies in therapeutic, diagnostic, and research applications.

RELATED APPLICATIONS

This is a continuation of U.S. Ser. No. 10/693,538, filed Oct. 23, 2003,which is a continuation of PCT/US02/11950, filed Apr. 17, 2002, whichclaims the benefit of U.S. Ser. No. 60/367,002, filed Mar. 22, 2002, andU.S. Ser. No. 60/301,091, filed Jun. 26, 2001, and U.S. Ser. No.60/293,020, filed on May 17, 2001, and U.S. Ser. No. 60/286,782, filedon Apr. 26, 2001. The entire disclosure of each of the aforesaid patentapplications are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the fields of genetics andcellular and molecular biology. More particularly, the invention relatesto antibodies which bind to and modulate the signaling of Cripto, kitscomprising such antibodies, and methods which use the antibodies.

BACKGROUND OF THE INVENTION

Cripto is a cell surface protein of 188 amino acid residuesserendipitously isolated in a cDNA screen of a human embryonic carcinomalibrary (Ciccodicola et al., 1989, EMBO J., vol. 8, no. 7, pp.1987-1991). The Cripto protein has at least two notable domains: acysteine-rich domain, and a domain first characterized as similar to thedomain found in the epidermal growth factor (EGF) family. Cripto wasoriginally classified as a member of the EGF family (Ciccodicola et al.,supra); however, subsequent analysis showed that Cripto did not bind anyof the known EGF receptors and its EGF-like domain was actuallydivergent from the EGF family (Bianco et al., 1999, J. Biol. Chem.,274:8624-8629).

The Cripto signaling pathway has remained elusive despite continuedinvestigation, with the literature supporting activation of severaldifferent pathways, including a MAP kinase pathway (DeSantis et al.,1997, Cell Growth Differ., 8:1257-1266; Kannan et al., 1997, J. Biol.Chem., 272:3330-3335), the TGF-β pathway (Gritsman et al., 1999,Development, 127:921-932; Schier et al., 2000, Nature, 403:385-389),possible interactions with the Wnt pathway (Salomon et al., Endocr RelatCancer. 2000 December; 7(4):199-226; and cross talk with the EGF pathway(Bianco et al., 1999, J. Biol. Chem., 274:8624-8629).

U.S. Pat. No. 5,256,643 and two divisional applications related thereto(U.S. Pat. Nos. 5,654,140 and 5,792,616), disclose a human Cripto gene,Cripto protein, and antibodies to Cripto.

U.S. Pat. No. 5,264,557 and three divisional applications relatedthereto (U.S. Pat. Nos. 5,620,866, 5,650,285, and 5,854,399), disclose ahuman Cripto-related gene and protein. Also disclosed are antibodieswhich bind to the Cripto-related protein but do not cross react bybinding to the Cripto protein itself.

Cripto protein overexpression is associated with many tumor types(including but not limited to breast, testicular, colon, lung, ovary,bladder, uterine, cervical, pancreatic, and stomach), as demonstrated byimmunostaining of human tissue with rabbit polyclonal antibodies raisedagainst small cripto peptides. Panico et al., 1996, Int. J. Cancer, 65:51-56; Byrne et al., 1998, J Pathology, 185:108-111; De Angelis et al.,1999, Int J Oncology, 14:437-440. The art is therefore in need of meansof controlling, restricting, and/or preventing such overexpression,modulating Cripto signaling, and modulating the consequences of Criptoexpression (i.e., promotion and/or maintenance of cell transformation).

SUMMARY OF THE INVENTION

The present invention provides novel antibodies which specifically bindto Cripto, and methods of making and using such antibodies. Theinvention also provides antibodies which bind to Cripto, and modulateCripto signaling or protein interaction, e.g., an antibody which bindsto Cripto such that the signal resulting from a protein interaction withCripto is modulated downward. The invention also provides antibodieswhich bind to Cripto and block the interaction between Cripto and ALK4.The invention also provides antibodies which bind to Cripto and modulatetumor growth. The invention also provides antibodies which bind toCripto, modulate Cripto signaling and modulate tumor growth. Theinvention also provides antibodies which bind to Cripto, block theinteraction between Cripto and ALK4 and modulate tumor growth

In one aspect of the invention, the antibody of the present inventionspecifically binds to an epitope selected from the group of epitopes towhich antibodies A6C12.11, A6F8.6 (ATCC ACCESSION NO. PTA-3318),A7H1.19, A8F1.30, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A8H3.1 (ATCCACCESSION NO. PTA-3315), A8H3.2, A19A10.30, A10B2.18 (ATCC ACCESSION NO.PTA-3311), A27F6.1 (ATCC ACCESSION NO. PTA-3310), A40G12.8 (ATCCACCESSION NO. PTA-3316), A2D3.23, A7A10.29, A9G9.9, A15C12.10, A15E4.14,A17A2.16, A17C12.28, A17G12.1 (ATCC ACCESSION NO. PTA-3314), A17H6.1,A18B3.11 (ATCC ACCESSION NO. PTA-3312), A19E2.7, B3F6.17 (ATCC ACCESSIONNO. PTA-3319), B6G7.10 (ATCC ACCESSION NO. PTA-3313), B11H8.4 bind.

In another aspect of the invention, the antibody of the presentinvention specifically binds to an epitope in the ligand/receptorbinding domain of Cripto. Cripto can be selected from CR-1 (SEQ ID NO:1)or CR-3 (SEQ ID NO:2). In a more particular embodiment, antibodies thatspecifically binds to the epitope in the ligand/receptor binding domaininclude for example A6C12.11, A6F8.6 (ATCC ACCESSION NO. PTA-3318),A8G3.5 (ATCC ACCESSION NO. PTA-3317), A19A10.30, A8H3.1 (ATCC ACCESSIONNO. PTA-3315), A27F6.1 (ATCC ACCESSION NO. PTA-3310), A40G12.8 (ATCCACCESSION NO. PTA-3316), A17G12.1 (ATCC ACCESSION NO. PTA-3314),A18B3.11 (ATCC ACCESSION NO. PTA-3312) and B6G7.10 (ATCC ACCESSION NO.PTA-3313).

In one embodiment the epitope to which the antibodies of the presentinvention bind is in an EGF-like domain. Antibodies that specificallybind to the epitope in the EGF-like domain include but are not limitedto A40G12.8 (ATCC ACCESSION NO. PTA-3316), A8H3.1 (ATCC ACCESSION NO.PTA-3315), A27F6.1 (ATCC ACCESSION NO. PTA-3310), B6G7.10 (ATCCACCESSION NO. PTA-3313), A17G12.1 (ATCC ACCESSION NO. PTA-3314) andA18B3.11 (ATCC ACCESSION NO. PTA-3312).

In another embodiment the epitope to which the antibodies of the presentinvention bind is in a cys-rich domain. Antibodies that specificallybind to the epitope in the cys-rich domain include but are not limitedto A19A10.30, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A6F8.6 (ATCCACCESSION NO. PTA-3318) and A6C12.11.

In another embodiment the epitope to which the antibodies of the presentinvention bind is in the domain spanning amino acid residues 46-62 ofCripto. Antibodies that specifically bind to the epitope in the domainspanning amino acid residues 46-62 of Cripto include but are not limitedto A10B2.18 (ATCC ACCESSION NO. PTA-3311), B3F6.17 (ATCC ACCESSION NO.PTA-3319) and A17A2.16.

The present inventions also contemplate antibodies which bindsspecifically to Cripto and are capable of modulating Cripto signaling.Antibodies that bind specifically to Cripto and are capable ofmodulating Cripto signaling, include but are not limited to, A40G12.8(ATCC ACCESSION NO. PTA-3316), A8H3.1 (ATCC ACCESSION NO. PTA-3315),A27F6.1 (ATCC ACCESSION NO. PTA-3310), and A6C12.11. In one embodimentthe antibodies of the present invention which binds specifically toCripto and are capable of modulating Cripto signaling bind to an epitopein an EGF-like domain or a cys-rich domain of Cripto.

The present inventions also contemplate antibodies which bindsspecifically to Cripto and blocks the interaction between Cripto andALK4. Antibodies that bind specifically to Cripto and are capable ofblocking the interaction between Cripto and ALK4, include but are notlimited to, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A6F8.6 (ATCC ACCESSIONNO. PTA-3318) and A6C12.11. In one embodiment the antibodies of thepresent invention which binds specifically to Cripto and are capable ofblocking the interaction between Cripto and ALK4 bind to an epitope inan EGF-like domain or a cys-rich domain of Cripto.

In another aspect, the present invention contemplates antibodies whichbind specifically to Cripto and are capable of modulating tumor growth.Antibodies that specifically bind to Cripto and are capable ofmodulating tumor growth include but are not limited to, A27F6.1 (ATCCACCESSION NO. PTA-3310), B6G7.10 (ATCC ACCESSION NO. PTA-3313) andA8G3.5 (ATCC ACCESSION NO. PTA-3317).

In one embodiment the antibodies of the present invention which bindspecifically to Cripto and are capable of modulating tumor growth bindto an epitope in an EGF-like domain or a cys-rich domain of Cripto.

In yet another aspect, the present invention contemplates antibodieswhich bind specifically to Cripto, which are capable of modulatingCripto signaling, and which are capable of modulating tumor growth.Antibodies that specifically bind to Cripto, which are capable ofmodulating Cripto signaling, and which are capable of modulating tumorgrowth, include but are not limited to A27F6.1 (ATCC ACCESSION NO.PTA-3310).

In one embodiment the antibodies of the present invention which bindspecifically to Cripto, which are capable of modulating Criptosignaling, and which are capable of modulating tumor growth bind to anepitope in an EGF-like domain or a cys-rich domain of Cripto.

In yet another aspect, the present invention contemplates antibodieswhich bind specifically to Cripto, which are capable of blocking theinteraction between Cripto and ALK4, and which are capable of modulatingtumor growth. Antibodies that specifically bind to Cripto, which arecapable of blocking the interaction between Cripto and ALK4, and whichare capable of modulating tumor growth, include but are not limited toA8G3.5 (ATCC ACCESSION NO. PTA-3317).

In another embodiment, the present invention provides an antibodyproduced by a hybridoma selected from the group consisting of A6F8.6(ATCC Accession No. PTA-3318), A8G3.5 (ATCC Accession No. PTA-3317),A8H3.1 (ATCC Accession No. PTA-3315), A10B2.18 (ATCC Accession No.PTA-3311), A27F6.1 (ATCC Accession No. PTA-3310), A40G12.8 (ATCCAccession No. PTA-3316), A17G12.1 (ATCC Accession No. PTA-3314),A18B3.11 (ATCC Accession No. PTA-3312), B3F6.17 (ATCC Accession No.PTA-3319), and B6G7.10 (ATCC Accession No. PTA-3313).

The antibodies of the present invention include but are not limited tomonoclonal, polyclonal, humanized, chimeric and human antibodies.

The present invention also provides for a composition for administrationto a subject having a tumor that expresses Cripto comprising at leastone of the antibodies described above. In a more particular embodimentthe subject is human. The composition may include a pharmaceuticallyacceptable excipient. The antibodies described above can be conjugatedto a chemotherapeutic agent or be provided in combination with anonconjugated chemotherapeutic.

Contemplated in another aspect of the invention are methods ofmodulating growth of tumor cells in vitro in a sample comprising thestep of adding to the sample the compositions described above.

Also contemplated are methods of modulating growth of tumor cells invivo in a subject comprising the step of administering to the subject aneffective amount of the compositions described above. In a particularembodiment the subject is human.

Another aspect of the invention are methods of treating subjects havinga tumor that over-expresses Cripto comprising administering to thesubject the compositions described above in an effective amount.Compositions for administration may include pharmaceutically acceptableexcipients, antibodies conjugated to chemotherapeutic agents andantibodies administered in combination with nonconjugatedchemotherapeutic agents

The methods of the present invention are particularly useful inmodulating growth of tumor cells and/or treating a subject (i.e. ahuman) having a tumor where the tumor cell is selected from breast,testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic,and stomach tumor cells.

In yet another embodiment, the present invention contemplates methods ofdetermining whether a tissue expresses Cripto, comprising the step ofanalyzing tissue from the subject in an immunoassay using any of theantibodies described above. Also contemplated are methods of determiningwhether a cell line overexpresses Cripto, comprising the step ofanalyzing the cell line in an immunoassay using any of the antibodiesdescribed above.

These and other aspects of the invention are set forth in greater detailbelow in the Detailed Description of the Invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the response of the human testicular carcinoma cell lineNCCIT to the monoclonal antibody A8G3.4, an antibody that binds thecys-rich domain of Cripo, in an in vivo xenograft tumor model in mice.

FIG. 2 depicts the response of the human testicular carcinoma cell lineNCCIT to the monoclonal antibody A27F6.1, an antibody that binds to theEGF-like domain of Cripto, in an in vivo xenograft tumor model in mice.

FIG. 3 depicts the results of flow cytometry analysis demonstrating thatCripto-specific monoclonal antibodies interfere with the binding ofCripto to the activin type I receptor, Alk4.

DETAILED DESCRIPTION OF THE INVENTION

Antibodies that specifically bind to Cripto and their uses formodulating Cripto signaling or protein interaction, and/or block theinteraction between Cripto and ALK4, and/or modulate the growth of tumorcells have been discovered. Various classes of antibodies thatspecifically bind to Cripto have been discovered, including, forexample, antibodies that specifically bind to an epitope in theligand/receptor binding domain of either a native Cripto protein or adenatured form of Cripto; antibodies that bind an EGF-like domain, acys-rich domain, or a peptide (e.g., from about 3 to about 20 aminoacids) from the region comprising amino acid residues 46 to 150;antibodies that bind Cripto and modulate Cripto signalling; antibodiesthat bind Cripto and modulate tumor cell growth; and antibodies thatbind to Cripto, modulate Cripto signaling, and modulate tumor cellgrowth. These antibodies are selected using conventional in vitro assaysfor selecting antibodies which bind the ligand/receptor binding domain,modulate Cripto signaling, or modulate tumor cell growth.

The methods of this invention are useful in the therapy of malignant orbenign tumors of mammals where the growth rate of the tumor (which is anabnormal rate for the normal tissue) is at least partially dependentupon Cripto. Abnormal growth rate is a rate of growth which is in excessof that required for normal homeostasis and is in excess of that fornormal tissues of the same origin.

Definitions

Various definitions are made throughout this document. Most words havethe meaning that would be attributed to those words by one skilled inthe art. Words specifically defined either below or elsewhere in thisdocument have the meaning provided in the context of the presentinvention as a whole and as are typically understood by those skilled inthe art.

As used herein, the term “region” means a physically contiguous portionof the primary structure of a biomolecule. In the case of proteins, aregion is defined by a contiguous portion of the amino acid sequence ofthat protein.

As used herein, the term “domain” refers to a structural part of abiomolecule that contributes to a known or suspected function of thebiomolecule. Domains may be co-extensive with regions or portionsthereof; domains may also incorporate a portion of a biomolecule that isdistinct from a particular region, in addition to all or part of thatregion. Examples of protein domains include, but are not limited to theextracellular domain (spans from about residue 31 to about residue 188of Cripto, including Cripto, CR-1 (SEQ ID NO: 1) and CR-3 (SEQ ID NO:2))and transmembrane domain (spans from about residue 169 to about residue188 of Cripto, including Cripto, CR-1 (SEQ ID NO: 1) and CR-3 (SEQ IDNO:2)). A ligand/receptor binding domain of the Cripto protein spansfrom about residue 75 to about residue 150 of Cripto, including Cripto,CR-1 (SEQ ID NO: 1) and CR-3 (SEQ ID NO:2) and includes the EGF-likedomain of Cripto, which spans, for example, from about residue 75 toabout residue 112 of Cripto, including Cripto, CR-1 (SEQ ID NO: 1) andCR-3 (SEQ ID NO:2) and the cysteine-rich domain of Cripto, which spans,for example, from about residue 114 to about residue 150 of Cripto,including Cripto, CR-1 (SEQ ID NO: 1) and CR-3 (SEQ ID NO:2). Forexample, many monoclonal antibodies of the present invention have beenidentified as binding to the EGF-like or cys-rich domains. Additionallymonoclonal antibody A10B2.18 (ATCC ACCESSION NO. PTA-3311), B3F6.17(ATCC ACCESSION NO. PTA-3319) and A17A2.16 have been identified asbinding to an epitope formed in a domain in the region spanning aminoacid residues 46-62, upstream of the EGF-like domain. See Example 3below. An epitope in the ligand/receptor binding domain is an epitope,whether formed in the conformational native Cripto protein, or thedenatured Cripto protein, to which antibodies may bind.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab, Fab′, F(ab)₂, and other fragments thereof.Complete, intact antibodies include, but are not limited to, monoclonalantibodies such as murine monoclonal antibodies, polyclonal antibodies,chimeric antibodies, human antibodies, and humanized antibodies. Variousforms of antibodies may be produced using standard recombinant DNAtechniques (Winter and Milstein, Nature 349: 293-99, 1991). For example,“chimeric” antibodies may be constructed, in which the antigen bindingdomain from an animal antibody is linked to a human constant domain (anantibody derived initially from a nonhuman mammal in which recombinantDNA technology has been used to replace all or part of the hinge andconstant regions of the heavy chain and/or the constant region of thelight chain, with corresponding regions from a human immunoglobulinlight chain or heavy chain) (see, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. 81: 6851-55, 1984).Chimeric antibodies reduce the immunogenic responses elicited by animalantibodies when used in human clinical treatments.

In addition, recombinant “humanized” antibodies may be synthesized.Humanized antibodies are antibodies initially derived from a nonhumanmammal in which recombinant DNA technology has been used to substitutesome or all of the amino acids not required for antigen binding withamino acids from corresponding regions of a human immunoglobulin lightor heavy chain. That is, they are chimeras comprising mostly humanimmunoglobulin sequences into which the regions responsible for specificantigen-binding have been inserted (see, e.g., PCT patent application WO94/04679). Animals are immunized with the desired antigen, thecorresponding antibodies are isolated and the portion of the variableregion sequences responsible for specific antigen binding are removed.The animal-derived antigen binding regions are then cloned into theappropriate position of the human antibody genes in which the antigenbinding regions have been deleted. Humanized antibodies minimize the useof heterologous (inter-species) sequences in antibodies for use in humantherapies, and are less likely to elicit unwanted immune responses.Primatized antibodies can be produced similarly.

Another embodiment of the invention includes the use of humanantibodies, which can be produced in nonhuman animals, such astransgenic animals harboring one or more human immunoglobulintransgenes. Such animals may be used as a source for splenocytes forproducing hybridomas, as is described in U.S. Pat. No. 5,569,825.

Antibody fragments and univalent antibodies may also be used in themethods and compositions of this invention. Univalent antibodiescomprise a heavy chain/light chain dimer bound to the Fc (or stem)region of a second heavy chain. “Fab region” refers to those portions ofthe chains which are roughly equivalent, or analogous, to the sequenceswhich comprise the Y branch portions of the heavy chain and to the lightchain in its entirety, and which collectively (in aggregates) have beenshown to exhibit antibody activity. A Fab protein includes aggregates ofone heavy and one light chain (commonly known as Fab′), as well astetramers which correspond to the two branch segments of the antibody Y,(commonly known as F(ab)2), whether any of the above are covalently ornon-covalently aggregated, so long as the aggregation is capable ofspecifically reacting with a particular antigen or antigen family.

Any of the antibodies of the invention may optionally be conjugated to achemotherapeutic, as defined below.

As used herein, the term “binding” means the physical or chemicalinteraction between two proteins or compounds or associated proteins orcompounds or combinations thereof, including the interaction between anantibody and a protein. Binding includes ionic, non-ionic, hydrogenbonds, Van der Waals, hydrophobic interactions, etc. The physicalinteraction, the binding, can be either direct or indirect, indirectbeing through or due to the effects of another protein or compound.Direct binding refers to interactions that do not take place through ordue to the effect of another protein or compound but instead are withoutother substantial chemical intermediates. Binding may be detected inmany different manners. Methods of detecting binding are well-known tothose of skill in the art.

As used herein, “an antibody capable of internalizing Cripto” means anantibody which enters the cell while removing Cripto from the cellsurface. One can screen for Cripto antibodies which are capable ofinternalizing Cripto by using fluorescent labeled Cripto monoclonalantibodies. In order to determine which antibodies internalize into theCripto positive cells one can assay for the uptake of the fluorescentsignal of the antibodies into the cells by viewing the cells under afluorescent and/or confocal microscope. Those antibodies that getinternalized will be seen as fluorescent signals in the cytoplasmic andor cellular vesicles. Non-limiting examples of Cripto antibodies capableof internalizing Cripto include A27F6.1 and B3F6.17.

As used herein, the term “compound” means any identifiable chemical ormolecule, including, but not limited to, ion, atom, small molecule,peptide, protein, sugar, nucleotide, or nucleic acid, and such compoundcan be natural or synthetic.

As used herein, the terms “modulates” or “modifies” means an increase ordecrease in the amount, quality, or effect of a particular activity orprotein.

As used herein, the term “modulate Cripto signaling” means an increaseor decrease in the amount, quality, or effect of Cripto activity, byabout 5%, preferably 10%, more preferably 20%, more preferably 30%, morepreferably 40%, more preferably 50%, more preferably 60%, morepreferably 70%, more preferably 80%, more preferably 90%, and mostpreferably 100%. Activity may be measured by assays known in the art,such as the null cell assay shown in Example 3. In another embodiment,protein interaction between Cripto and another protein is similarlymodulated downward via binding of the antibodies of the invention.

As used herein, the term “blocking the interaction between Cripto andALK 4” means an increase or decrease in the interaction, i.e. binding,between Cripto and ALK4, by about 5%, preferably 10%, more preferably20%, more preferably 30%, more preferably 40%, more preferably 50%, morepreferably 60%, more preferably 70%, more preferably 80%, morepreferably 90%, and most preferably 100%. Activity may be measured byassays known in the art, such as the binding assay shown in Example 8.

As used herein, the term “modulate growth of tumor cells in vitro” meansan increase or decrease in the number of tumor cells, in vitro, by about5%, preferably 10%, more preferably 20%, more preferably 30%, morepreferably 40%, more preferably 50%, more preferably 60%, morepreferably 70%, more preferably 80%, more preferably 90%, and mostpreferably 100%. In vitro modulation of tumor cell growth may bemeasured by assays known in the art, such as the GEO cell soft agarassay shown in Example 4.

As used herein, the term “modulate growth of tumor cells in vivo” meansan increase or decrease in the number of tumor cells, in vivo, by about5%, preferably 10%, more preferably 20%, more preferably 30%, morepreferably 40%, more preferably 50%, more preferably 60%, morepreferably 70%, more preferably 80%, more preferably 90%, and mostpreferably 100%. In vivo modulation of tumor cell growth may be measuredby assays known in the art, such as the one shown in Example 5.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops an abnormal condition.

The term “treating” refers to having a therapeutic effect and at leastpartially alleviating or abrogating an abnormal condition in theorganism. Treating includes maintenance of inhibited tumor growth, andinduction of remission.

The term “therapeutic effect” refers to the inhibition of an abnormalcondition. A therapeutic effect relieves to some extent one or more ofthe symptoms of the abnormal condition. In reference to the treatment ofabnormal conditions, a therapeutic effect can refer to one or more ofthe following: (a) an increase or decrease in the proliferation, growth,and/or differentiation of cells; (b) inhibition (i.e., slowing orstopping) or promotion of cell death; (c) inhibition of degeneration;(d) relieving to some extent one or more of the symptoms associated withthe abnormal condition; and (e) enhancing the function of a populationof cells. Compounds demonstrating efficacy against abnormal conditionscan be identified as described herein.

The term “administering” relates to a method of incorporating a compoundinto cells or tissues of an organism. The abnormal condition can beprevented or treated when the cells or tissues of the organism existwithin the organism or outside of the organism. Cells existing outsidethe organism can be maintained or grown in cell culture dishes, or inanother organism. For cells harbored within the organism, manytechniques exist in the art to administer compounds, including (but notlimited to) oral, parenteral, dermal, injection, and aerosolapplications. For cells outside of the organism, multiple techniquesexist in the art to administer the compounds, including (but not limitedto) cell microinjection techniques, transformation techniques andcarrier techniques. Administration may be accomplished by the many modesknown in the art, e.g., oral, intravenous, intraperitoneal,intramuscular, and the like. When used in in vivo therapy, theantibodies of the subject invention are administered to a patient ineffective amounts. As used herein an “effective amount” is an amountsufficient to effect beneficial or desired clinical results (i.e.,amounts that eliminate or reduce the patient's tumor burden). Aneffective amount can be administered in one or more administrations. Forpurposes of this invention, an effective amount of the antibodies of thepresent invention is an amount of the antibodies that is sufficient toameliorate, stabilize, or delay the development of the Cripto-associateddisease state, particularly Cripto-associated tumors. Detection andmeasurement of these indicators of efficacy are discussed below. Anexample of a typical treatment regime includes administering byintravenous infusion to the subject antibodies of the invention on aweekly schedule, at a dose of about 2-5 mg/kg. The antibodies areadministered in an outpatient chemoinfusion unit, unless the patientrequires hospitalization. Other administration regimes known in the artare also contemplated.

The abnormal condition can also be prevented or treated by administeringan antibody of the invention to a group of cells having an aberration ina signal transduction pathway to an organism. The effect ofadministering a compound on organism function can then be monitored. Theorganism is preferably a human.

“Cripto overexpression” is intended to mean the expression of Cripto bya tissue which expression is greater than the Cripto expression ofadjacent normal tissue in a statistically significant amount.

“Chemotherapeutics” refers to any agents identified in the art as havingtherapeutic effect on the inhibition of tumor growth, maintenance ofinhibited tumor growth, and/or induction of remission, such as naturalcompounds, synthetic compounds, proteins, modified proteins, andradioactive compounds. Chemotherapeutic agents contemplated herewithinclude agents that can be conjugated to the antibodies of the presentinvention or alternatively agents that can be used in combination withthe antibodies of the present invention without being conjugated to theantibody. Exemplary chemotherapeutics that can be conjugated to theantibodies of the present invention include, but are not limited toradioconjugates (90Y, 131I, 99mTc, 111In, 186Rh, et al.),tumor-activated prodrugs (maytansinoids, CC-1065 analogs, clicheamicinderivatives, anthracyclines, vinca alkaloids, et al.), ricin, diptheriatoxin, pseudomonas exotoxin.

Chemotherapeutic agents may be used in combination with the antibodiesof the invention, rather than being conjugated thereto (i.e.nonconjugated chemotherapeutics), include, but are not limited to thefollowing: platinums (i.e. cis platinum), anthracyclines, nucleosideanalogs (purine and pyrimidine), taxanes, camptothecins,epipodophyllotoxins, DNA alkylating agents, folate antagonists, vincaalkaloids, ribonucleotide reductase inhibitors, estrogen inhibitors,progesterone inhibitors, androgen inhibitors, aromatase inhibitors,interferons, interleukins, monoclonal antibodies, taxol, camptosar,adriamycin (dox), 5-FU and gemcitabine. Such chemotherapeutics may beemployed in the practice of the invention in combination with theantibodies of the invention by coadministration of the antibody and thenonconjugated chemotherapeutic.

“Pharmaceutically acceptable carrier or excipient” refers tobiologically inert compounds known in the art and employed in theadministration of the antibodies of the invention. Acceptable carriersare well known in the art and are described, for example, in Remington'sPharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., 1990.Acceptable carriers can include biocompatible, inert or bioabsorbablesalts, buffering agents, oligo- or polysaccharides, polymers,viscoelastic compound such as hyaluronic acid, viscosity-improvingagents, preservatives, and the like.

A “subject” refers to vertebrates, particularly members of a mammalianspecies, and includes but is not limited to domestic animals, sportsanimals, and primates, including humans.

Antibodies of the Invention

The antibodies of the invention specifically bind to Cripto: As usedherein, Cripto includes the CR-1 Cripto protein, the CR-3 Criptoprotein, and fragments thereof. Such fragments may be entire domains,such as the extracellular or intracellular domains, the EGF-like domain,the cys-rich domain, the receptor binding domain, and the like. Suchfragments may also include contiguous and noncontiguous epitopes in anydomain of the Cripto protein.

The 188 amino acid sequence for CR-1 is as follows [SEQ ID NO: 1]:

MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDSIWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPSFYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCDGLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY

The 188 amino acid sequence for CR-3 is as follows [SEQ ID NO: 2]:

MDCRKMVRFSYSVIWIMAISKAFELGLVAGLGHQEFARPSRGDLAFRDDSIWPQEEPAIRPRSSQRVLPMGIQHSKELNRTCCLNGGTCMLESFCACPPSFYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCDGLVMDEHLVASRTPELPPSARTTTFMLAGICLSIQSYY

In a one embodiment, the antibodies of the invention bind to an epitopein the EGF-like domain of Cripto. The EGF-like domain spans from aboutamino acid residue 75 to about amino acid residue 112 of the matureCripto protein. Epitopes in the EGF-like domain may comprise linear ornonlinear spans of amino acid residues. Example of linear epitopescontemplated include but are not limited to about residues 75-85, 80-90,85-95, 90-100, 95-105, 100-110, or 105-112. In one embodiment, theepitope in the EGF domain is an epitope formed in the conformationalnative Cripto protein versus a denatured Cripto protein.

In another embodiment, the antibodies of the invention bind to anepitope in the cys-rich domain of Cripto. The cys-rich domain spans fromabout amino acid residue 114 to about amino acid residue 150 of themature Cripto protein. Epitopes in the cys-rich domain may compriselinear or nonlinear spans of amino acid residues. Example of linearepitopes contemplated include but are not limited to about residues114-125, 120-130, 125-135, 130-140, 135-145, or 140-150. In oneembodiment, the epitope in the cys-rich domain is an epitope formed inthe conformational native Cripto protein versus a denatured Criptoprotein

Once antibodies are generated, binding of the antibodies to Cripto maybe assayed using standard techniques known in the art, such as ELISA,while the presence of Cripto on a cell surface may be assayed using flowcytometry (FACS), as shown in Example 2. Any other techniques ofmeasuring such binding may alternatively be used.

The present invention provides antibodies (e.g., monoclonal andpolyclonal antibodies, single chain antibodies, chimeric antibodies,bifunctional/bispecific antibodies, humanized antibodies, humanantibodies, and complementary determining region (CDR)-graftedantibodies, including compounds which include CDR sequences whichspecifically recognize a polypeptide of the invention) specific forCripto or fragments thereof. Antibody fragments, including Fab, Fab′,F(ab′)₂, and F_(v), are also provided by the invention. The terms“specific” and “selective,” when used to describe binding of theantibodies of the invention, indicates that the variable regions of theantibodies of the invention recognize and bind Cripto polypeptides. Itwill be understood that specific antibodies of the invention may alsointeract with other proteins (for example, S. aureus protein A or otherantibodies in ELISA techniques) through interactions with sequencesoutside the variable region of the antibodies, and, in particular, inthe constant region of the molecule. Screening assays to determinebinding specificity of an antibody of the invention (i.e. antibodiesthat specifically bind to an epitope the ligand/receptor binding domainand the domain spanning amino acid residues 46-62) are well known androutinely practiced in the art. For a comprehensive discussion of suchassays, see Harlow et al. (Eds.), Antibodies A Laboratory Manual; ColdSpring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.Antibodies that recognize and bind fragments of Cripto protein are alsocontemplated, provided that the antibodies are specific for Criptopolypeptides. Antibodies of the invention can be produced using anymethod well known and routinely practiced in the art.

In one embodiment, the invention provides an antibody that specificallybinds to an epitope in the ligand/receptor binding domain of Cripto.Antibody specificity is described in greater detail below. However, itshould be emphasized that antibodies that can be generated from otherpolypeptides that have previously been described in the literature andthat are capable of fortuitously cross-reacting with Cripto (e.g., dueto the fortuitous existence of a similar epitope in both polypeptides)are considered “cross-reactive” antibodies. Such cross-reactiveantibodies are not antibodies that are “specific” for Cripto. Thedetermination of whether an antibody specifically binds to an epitope ofCripto is made using any of several assays, such as Western blottingassays, that are well known in the art. For identifying cells thatexpress Cripto and also for modulating Cripto ligand/receptor bindingactivity, antibodies that specifically bind to an extracellular epitopeof the Cripto protein (i.e., portions of the Cripto protein foundoutside the cell) are particularly useful.

In one embodiment, the invention provides a cell-free compositioncomprising polyclonal antibodies, wherein at least one of the antibodiesis an antibody of the invention specific for Cripto. Antisera isolatedfrom an animal is an exemplary composition, as is a compositioncomprising an antibody fraction of an antisera that has been resuspendedin water or in another diluent, excipient, or carrier.

In another embodiment, the invention provides monoclonal antibodies.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Further in contrast to polyclonal preparationswhich typically include different antibodies directed against differentepitopes, each monoclonal antibody is directed against a singledeterminant on the antigen. Monoclonal antibodies are useful to improveselectivity and specificity of diagnostic and analytical assay methodsusing antigen-antibody binding. Another advantage of monoclonalantibodies is that they are synthesized by a hybridoma culture,uncontaminated by other immunoglobulins. Hybridomas that produce suchantibodies are also intended as aspects of the invention.

In still another related embodiment, the invention provides ananti-idiotypic antibody specific for an antibody that is specific forCripto. For a more detailed discussion of anti-idiotypic antibodies,see, e.g., U.S. Pat. Nos. 6,063,379 and 5,780,029.

It is well known that antibodies contain relatively small antigenbinding domains that can be isolated chemically or by recombinanttechniques. Such domains are useful Cripto binding molecules themselves,and also may be reintroduced into human antibodies, or fused to achemotherapeutic or polypeptide. Thus, in still another embodiment, theinvention provides a polypeptide comprising a fragment of aCripto-specific antibody, wherein the fragment and associated molecule,if any, bind to the Cripto. By way of non-limiting example, theinvention provides polypeptides that are single chain antibodies andCDR-grafted antibodies. For a more detailed discussion of CDR-graftedantibodies, see, e.g., U.S. Pat. No. 5,859,205.

In another embodiment, non-human antibodies may be humanized by any ofthe methods known in the art. Humanized antibodies are useful for invivo therapeutic applications. In addition, recombinant “humanized”antibodies may be synthesized. Humanized antibodies are antibodiesinitially derived from a nonhuman mammal in which recombinant DNAtechnology has been used to substitute some or all of the amino acidsnot required for antigen binding with amino acids from correspondingregions of a human immunoglobulin light or heavy chain. That is, theyare chimeras comprising mostly human immunoglobulin sequences into whichthe regions responsible for specific antigen-binding have been inserted(see, e.g., PCT patent application WO 94/04679). Animals are immunizedwith the desired antigen, the corresponding antibodies are isolated andthe portion of the variable region sequences responsible for specificantigen binding are removed. The animal-derived antigen binding regionsare then cloned into the appropriate position of the human antibodygenes in which the antigen binding regions have been deleted. Humanizedantibodies minimize the use of heterologous (inter-species) sequences inantibodies for use in human therapies, and are less likely to elicitunwanted immune responses. Primatized antibodies can be producedsimilarly using primate (e.g., rhesus, baboon and chimpanzee) antibodygenes. Further changes can then be introduced into the antibodyframework to modulate affinity or immunogenicity. See, e.g., U.S. Pat.Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370.

Another embodiment of the invention includes the use of humanantibodies, which can be produced in nonhuman animals, such astransgenic animals harboring one or more human immunoglobulintransgenes. Such animals may be used as a source for splenocytes forproducing hybridomas, as is described in U.S. Pat. No. 5,569,825,WO00076310, WO00058499 and WO00037504 and incorporated by referenceherein.

Signal Modulation

In another embodiment, the antibodies of the invention bind to Cripto,and modulate Cripto signaling or Cripto-protein interactions.Over-expression of Cripto activity can lead to a de-differentiated statepromoting mesenchymal cell characteristics, increased proliferation, andcell migration (Salomon et al., BioEssays 21: 61-70, 1999; Ciardiello etal., Oncogene 9: 291-298, 1994; and Baldassarre et al., Int. J. Cancer66:538-543, 1996), phenotypes associated with cell transformation seenin neoplasia.

One method of testing the activity of anti-Cripto antibodies and theirability to modulate Cripto signaling is with an F9-Cripto knock-out (KO)cell line (Minchiotti at al., Mech. Dev. 90: 133-142, 2000). Criptostimulates smad2 phosphorylation and the transcription factor FAST inXenopus embryos, and the activity of the transcription factor FAST canbe monitored by measuring the luciferase activity from a FAST regulatoryelement-luciferase reporter gene (Saijoh et al., Mol. Cell. 5:35-47,2000). F9-Cripto KO cells are deleted for the Cripto gene and are thusnull for Cripto and Cripto-dependent signaling (Minchiotti at al., Mech.Dev. 90: 133-142, 2000). Cripto signaling can be assessed in the F9Cripto KO cells by transfecting in Cripto, FAST, and the FAST regulatoryelement-luciferase gene construct. No Cripto dependent FAST luciferaseactivity will be seen in these cell lines unless Cripto cDNA, and FASTcDNA is transfected into them. Antibodies capable of blockingCripto-dependent Nodal signaling are antibodies that block Criptosignaling function.

Other assays capable of measuring the activity of Cripto can be employedby those of skill in the art, such as a growth in soft agar assay (seeExample 4 below). The ability of cells to grow in soft agar isassociated with cell transformation and the assay is a classical invitro assay for measuring inhibition of tumor cell growth. Other assaysuseful in determining inhibition of activity include in vitro assays onplastic, and the like.

Therapeutic Uses

Antibodies of the invention are also useful for, therapeutic purposes,such as modulation of tumor cell growth, diagnostic purposes to detector quantitate Cripto, and purification of Cripto.

In one embodiment of the invention, antibodies are provided which arecapable of binding specifically to Cripto and which modulate growth oftumor cells in a patient. In one embodiment, the tumor cells aretesticular, breast, testicular, colon, lung, ovary, bladder, uterine,cervical, pancreatic, and stomach tumor cells.

In another embodiment, antibodies are provided which are capable ofbinding specifically to Cripto and which modulate growth of tumor cellswhich overexpress Cripto. In one embodiment, the tumor cells are celllines which overexpress Cripto, such as cell lines derived from breast,testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic,and stomach cancer.

Anti-Cripto antibodies may be screened for in vivo activity as potentialanticancer agents following standard protocols used by those of skill inthe art, as illustrated in Example 4 below. Example of such protocolsare outlined by the National Cancer Institute (NCI) in their “in vivocancer models screening” protocols, NIH publication number 84-2635(February 1984).

In another embodiment of the invention, the antibodies of the inventionare used to treat a patient having a cancerous tumor.

The antibodies of the present invention can be combined with apharmaceutically acceptable excipient and administered in atherapeutically effective dose to the patient. For a discussion ofmethods of inhibiting growth of tumors, see, e.g., U.S. Pat. No.6,165,464.

Also contemplated are methods of treating a subject suffering from adisorder associated with abnormal levels (i.e. elevated or depleted) ofCripto wherein the method comprises administering to the subject aneffective amount of an antibody that specifically binds to an epitope inthe ligand/receptor binding domain of Cripto, including but not limitedto where the epitope is in an EGF-like domain or a cys-rich domain ofCripto.

Also contemplated are methods of treating a subject suffering from adisorder associated with abnormal levels (i.e. elevated or depleted) ofCripto wherein the method comprises administering to the subject aneffective amount of an antibody which specifically forms a complex withCripto and is directed to the epitope to which an antibody selected fromthe group consisting of A6C12.11, A6F8.6 (ATCC ACCESSION NO. PTA-3318),A7H1.19, A8F1.30, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A8H3.1 (ATCCACCESSION NO. PTA-3315), A8H3.2, A19A10.30, A10B2.18 (ATCC ACCESSION NO.PTA-3311), A27F6.1 (ATCC ACCESSION NO. PTA-3310), A40G12.8 (ATCCACCESSION NO. PTA-3316), A2D3.23, A7A10.29, A9G9.9, A15C12.10, A15E4.14,A17A2.16, A17C12.28, A17G12.1 (ATCC ACCESSION NO. PTA-3314), A17H6.1,A18B3.11 (ATCC ACCESSION NO. PTA-3312), A19E2.7, B3F6.17 (ATCC ACCESSIONNO. PTA-3319), and B6G7.10 (ATCC ACCESSION NO. PTA-3313) is directed.

Diagnosis via detection of Cripto is readily accomplished throughstandard binding assays using the novel antibodies of the invention,allowing those of skill in the art to detect the presence of Criptospecifically in a wide variety of samples, cultures, and the like.

Kits comprising an antibody of the invention for any of the purposesdescribed herein are also comprehended. In general, a kit of theinvention also includes a control antigen for which the antibody isimmunospecific. Embodiments include kits comprising all reagents andinstructions for the use thereof.

Additional features of the invention will be apparent from the followingillustrative Examples.

EXAMPLES Example 1 Expression and Purification of Cripto

An expression plasmid designated pSGS480 was constructed by sub-cloninga cDNA encoding human Cripto amino acids residues 1 to 169 of Cripto[amino acids 1-169 of SEQ ID NO: 1], fused to human IgG₁ Fc domain(i.e., “CR(del C)-Fc”) into vector pEAG100. For a more detaileddescription of this vector, see copending U.S. Patent Application Ser.No. 60/233,148, filed Sep. 18, 2000. The vector pEAG1100 is a derivativeof GIBCO-BRL Life Technologies plasmid pCMV-Sport-betagal, the use ofwhich in CHO transient transfections was described by Schifferli et al.,1999, Focus 21: 16. It was made by removing the reporter genebeta-galactosidase NotI fragment from the plasmid pCMV-Sport-Betagal(catalog number 10586-014) as follows: The plasmid was digested withNotI and EcoRV, the 4.38 kb NotI vector backbone fragment wasgel-purified and ligated. Ligated DNA was transformed into competent E.coli DH5alpha. pEAG1100 was isolated as a plasmid containing the desiredrecombinant from an isolated single colony. The sequence of pEAG1100spanning the promoter, polylinker, and transcription termination signalwas confirmed.

Plasmid pSGS480 was transiently transfected into CHO cells and the cellswere grown at 28° C. for 7 days. The presence of CR(del C)-Fc protein inthese cells and the conditioned media was examined by Western blotanalysis. For Western blot analysis, conditioned media and cells fromCripto transfected cells were subjected to SDS-PAGE on 4-20% gradientgels under reducing conditions, transferred electrophoretically tonitrocellulose, and the Cripto fusion protein was detected with a rabbitpolyclonal antiserum raised against a Cripto 17-mer peptide (comprisingresidues 97-113 of SEQ ID NO: 1)-keyhole limpet hemocyanin conjugate.After centrifugation to remove the cells, Western blot analysis showedthat the CR(del C)-Fc protein was efficiently secreted into theconditioned media (supernatant). The supernatant was applied to aProtein A-Sepaharose (Pharmacia), and bound protein was eluted with 25mM sodium phosphate pH 2.8, 100 mM NaCl. The eluted protein wasneutralized with 0.5 M sodium phosphate at pH 8.6, and analyzed fortotal protein content from absorbance measurements at 240-340 nm, andfor purity by SDS-PAGE. The eluted protein was filtered through a 0.2micron filter, and stored at −70° C.

Example 2 Generation and Screening of Antibodies

The eluted CR(del C)-Fc protein is injected into mice, and standardhybridoma techniques known to those of skill in the art are used togenerate monoclonal antibodies.

A. Generation of Antibodies

Particularly, female Robertsonian mice (Jackson Labs) were immunizedintraperitoneally with 25 μg of purified human CR del C-Fc emulsifiedwith complete fruend's adjuvant (GibcoBRL #15721-012). They were boostedtwo times intraperitoneally with 25 μg of CR del C-Fc emulsified withincomplete freunds's adjuvant (GibcoBRL #15720-014) and once on ProteinA beads. The sera were screened and 3 weeks after the last boost, themouse with the best titer was boosted intraperitoneally with 50 μgsoluble CR del C-Fc three days before fusion. The mouse was boostedintravenously with 50 μg CR del C-Fc the day before fusion. The mousespleen cells were fused with FL653 myeloma cell at a Ispleen:6 myelomaratio and were plated at 100,000, 33,000 and 11,000 cells per well into96 well tissue culture plates in selection media. Wells positive forgrowth were screened by FACS and ELISA a week later. Two fusions wereperformed.

B. Screening of Antibodies

Supernatants resulting from the first or second fusion were screenedfirst on ELISA plates for recognition of Cripto del C and/or CriptoEGF-like domain proteins. A control fusion protein (LT-beta receptor-Fc)was coated on ELISA plates to discard monoclonal antibodies thatrecognized the human Fc epitope. The ELISA was performed as describedbelow in section C. In the first fusion, primary supernatants were alsoscreened for their ability to recognize cell surface Cripto protein onthe testicular tumor cell line, NCCIT by FACS. In the case of the secondfusion, the ability of supernatants to recognize Cripto on two tumorcell lines, NCCIT and the breast cancer line, DU4475 by FACs wasanalyzed. Secondary screens included testing the monoclonal antibodysupernatant's ability to recognize cell surface Cripto on a panel oftumor cell lines (see Tables 1 and 2 for results), ability of monoclonalantibodies to recognize human Cripto immunohistochemically on humanbreast and colon tumor tissue sections, ability of monoclonal antibodiesto block in Cripto-Nodal signalling assay, ability to block growth oftumor cell lines on plastic or in soft agar assays, and ability tointernalize cell surface Cripto.

C. ELISA

The ELISA assays were performed as follows:

Materials:

Plates: Costar high-binding Easy-wash 96 W plates (07-200-642)

2′ antibody: Pierce Gt anti-Ms IgG (H+L)-HRP (P131430)

Substrate: Pierce TMB Substrate Kit (34021)

Stop solution: 1N H2SO4

Buffers:

Binding buffer: 0.1 M NaHPO4 pH 9.0

Blocking buffer: PBS+10% Donor Calf Serum

Wash buffer: PBS+0.1% tween-20

Antigens CR-del-C-Fc and CR-EGF-fc, control hu IgG1 fusion protein werediluted in binding buffer to 500 ng/ml. 100 μl were added per well andincubated for 1 hr at 37° C. or overnight at 4° C. The liquid wasdecanted and the plate inverted and blotted until dry. 250 μl/wellblocking buffer was then added, followed by incubation for 30 min. at37° C. Again, the liquid was decanted and the plate inverted and blotteduntil dry. Supernatants were diluted 1:50 in wash buffer, and plated at50 μl/well, followed by incubation for 1 hour at room temperature.Plates were washed 3× vigorously with 250 μl/well wash buffer. Then 100μl/well 2′ antibody diluted in wash buffer at 1:10,000 was added,followed by incubation for 30 min. at room temperature. Plates were thenwashed 3× vigorously with 250 μl/well wash buffer, then substrate addedat 100 μl/well. Color was permitted to develop until sufficiently dark,then 100 μl/well stop solution was added and the plates read forabsorbance at 450 nm.

D. Flow Cytometry

Cripto positive cell lines may be used to assay the monoclonalantibodies for binding to Cripto using cell surface staining and flowcytometry as follows:

Release cells from T162 flasks with 2 ml PBS⁻ with 5 mM EDTA, 10 min.,37° C. Bring up to 20 ml with media with serum, pipetting up and downseveral times to unclump cells. Spin at 1200 rpm for 5 minutes. Washcells with 5-10 ml 4° C. PBS with 0.1% BSA (wash buffer). Spin at 1200rpm for 5 minutes. Resuspend at 4×10⁶-10⁷/ml in wash buffer. Keep onice.

Prepare antibodies for staining. Purified antibodies are diluted to 1-10μg/ml in wash buffer. Add 50 μl of cells to a 96-well Linbro V bottomedplate (ICN 7632105). Plate one well of cells for each control for eachcell line to be analyzed, including cells for no antibody, 20 antibodyonly, hybridoma media, positive control antibody supernatant, ifavailable, or purified, and an IgG subclass control (if using purifiedantibodies).

Plate one well of cells for each experimental sample for each cell lineto be analyzed. Spin plate, 1200 rpm for 5 minutes, using a table topcentrifuge at 4° C. Flick out buffer by inverting the plate and shakinguntil the liquid is substantially discarded. Add 40-50 μl of antibodies(or wash buffer for the no-antibody and 2° antibody-only control wells)to wells. Incubate at least 30 min.-1 hour at 4° C. Spin plate, 1200 rpmfor 5 minutes. Flick out antibody solutions. Wash wells twice with 200μl wash buffer per well, spinning after each wash. Flick out buffer.

Resuspend cells in each well in 50 μl of 1:200 dilution (in wash buffer)of R-PE tagged goat anti-mouse IgG, Fc Specific (Jackson ImmunoresearchLaboratories Cat# 115-116-071). Incubate 20 min, 4° C., in the dark. Add150 μl wash buffer to cells in each well. Spin plate at 1200 rpm for 5minutes. Wash once with 200 μl wash buffer per well. Resuspend cells in150 μl 1% PFA in PBS. Transfer contents of each well to separate tubes(5 ml Falcon polystyrene round bottomed tube-352052). Wrap tubes in tinfoil.

The contents of the tubes are then read by flow cytometry.

The results of a two screenings of monoclonal antibodies produced bythis method yielded the following results, summarized in Tables 1 and 2below, wherein the first column provides the designated names for thehybridoma subclones, the next two columns show the results of ELISAscreens, and the remaining columns show flow cytometry analysis resultson four cripto-positive cell lines. The results are given in units ofmean fluorescent index (MFI).

TABLE 1 Anti-Cripto Monoclonal Antibody Characterization ELISA ELISACripto ATCC Cripto EGFlike Hybridoma deposit delC domain DU4475 NCCITGEO HT3 Subclone no. Sups Sups MFI MFI MFI MFI Control- 0.06 0.07 ELISAControl- 14 9 37 18 MouseIg A6C12.11 2.21 0.07 11 35 29 8 A6F8.6PTA-3318 2.32 0.08 11 50 29 10 A7H1.19 2.14 0.09 14 34 27 12 A8F1.302.15 0.1 17 27 32 28 A8G3.5 PTA-3317 2.39 0.09 9 30 25 15 A8H3.1PTA-3315 2.4 1.7 9 44 23 10 A8H3.2 2.54 0.07 13 13 16 14 A19A10.30 2.020.09 9 40 20 10 A10B2.18 PTA-3311 2.36 0.07 40 63 100 43 A27F6.1PTA-3310 2.28 1.19 9 44 26 17 A40G12.8 PTA-3316 2.27 1.59 10 47 26 16

TABLE 2 Anti-Cripto Monoclonal Antibody Characterization ELISA ATCCELISA Cripto Hybridoma deposit Cripto EGFlike DU4475 NCCIT GEO HT3Subclone no. delC domain MFI MFI MFI MFI Control- 0.05 0.05 ELISAControl- 10 6 4 6 MouseIg A2D3.23 0.93 0.90 73 138 37 27 A7A10.29 1.370.07 75 83 33 83 A9G9.9 1.39 0.07 52 62 32 82 A15C12.10 1.42 0.06 46 5525 93 A15E4.14 1.38 0.06 50 63 23 95 A17A2.16 1.40 0.06 76 97 41 81A17C12.28 0.96 0.97 6 16 3 22 A17G12.1 PTA-3314 1.30 1.37 61 66 28 78A17H6.1 1.38 0.05 35 30 5 28 A18B3.11 PTA-3312 1.36 1.38 50 42 33 65A19E2.7 1.40 0.06 53 59 26 99 B3F6.17 PTA-3319 1.37 0.06 77 51 39 89B6G7.10 PTA-3313 1.38 1.40 28 22 22 56 B11H8.4 1.41 0.06 59 101 39 107B12C12.5 1.10 1.04 27 14 23 59 B15A2.6 1.40 0.06 36 44 22 59 C4A2.161.40 0.06 24 36 22 65

Example 3 Null Cell Assay for Inhibition of Cripto Signaling

The following describes an F9 Cripto null cell signaling assay used toassess inhibition of Cripto signaling.

Day 0 Coat 6 welled plates with 0.1% gelatin 2 ml/well at 37° C. for 15min.

Seed cells at 6×10⁵ F9 CRIPTO NULL cells per well.

Day 1 Transfection:

Each of the following samples is added to 300 μl OptiMem1 to yieldSolution A for each sample:

Sample 1: 0.5 μg (N₂)₇ luciferase FAST reporter cDNA plus 1.5 μg emptyvector cDNA.Sample 2: 0.5 μg (N₂)₇ luciferase, 0.5 μg FAST, and 1 μg empty vectorcDNAs.Sample 3: 0.5 μg (N₂)₇ luciferase, 0.5 μg Cripto ADD 0.5 FAST, and 40.5μg empty vector cDNAs.Sample 4: 0.5 μg (N₂)₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μgempty vector cDNAsSample 5: 0.5 μg (N₂)₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μgempty vector cDNAs.Sample 6: 0.5 μg (N₂)₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μgempty vector cDNAs.Sample 7: 0.5 μg (N₂)₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μgempty vector cDNAs.Sample 8: 0.5 μg (N₂)₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μgempty vector cDNAs.Sample 9: 0.5 μg (N₂)₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μgempty vector cDNAs.

Solution B comprises 30 μl of Lipofectamine plus 270 μl of OptiMem1.

For each sample, mix solution A and solution B together. Incubate 45minutes at room temperature. Rinse wells with 2 ml/well of OptiMem1.Aspirate just before next step.

Add 2.4 ml of OptiMem1 to each mixture of solutions A+B, mix, add 1.5ml/well to duplicate wells. Incubate 5 hours at 37° C. Add 1.5 ml/wellof DMEM+20% FCS, 2 mM Gln, P/S to wells which received samples 1-3. Addanti-Cripto antibodies as follows: Sample 4 wells: A27F6.1, 10 μg/ml;Sample 5 wells: A27F6.1, 2 μg/ml; Sample 6 wells: A40G12.8; 10 μg/ml,Sample 7 wells: A40G12.8 2 μg/ml; Sample 8 wells: A10B2.18, 10 μg/ml;Sample 9 wells: A10B2.18, 2 μg/ml.

Day 2 Remove media, wash cells with PBS, 2 ml/well. Add DMEM+0.5% FCS, 2mM Gln, P/S with the same amounts of Cripto antibodies as the previousday, to the same wells.

Day 3 Develop luciferase signal. Wash wells with PBS+Ca²⁺ and Mg²⁺, 2ml/well. Use LucLite kit, Packard cat# 6016911. Bring buffer andsubstrate to room temperature. Dim lights. Reconstitute substrate with10 ml of buffer. Dilute 1:1 with PBS+Ca²⁺ and Mg²⁺. Aspirate wells.Quickly add 250 μl of diluted substrate per well using a repeatpipettor. Swirl solution and transfer 200 μl to wells of a 96 welledwhite opaque bottom plate, Falcon 35-3296. Read plate on luminometerusing Winglow, exporting data to Excel.

The results of this assay are summarized below in Table 3.

TABLE 3 Cripto Signaling Assay: Inhibition with Anti-Cripto MonoclonalAntibodies Relative Luminescent cDNAs transfected Anti-Cripto AntibodyUnits (N₂)₇ luc none 123 (N₂)₇ luc, FAST none 259 (N₂)₇ luc, FAST,Cripto none 3091 (N₂)₇ luc, FAST, Cripto A27F6.1 10 μg/ml 1507 (N₂)₇luc, FAST, Cripto A27F6.1 2 μg/ml 2297 (N₂)₇ luc, FAST, Cripto A40G12.810 μg/ml 1213 (N₂)₇ luc, FAST, Cripto A40G12.8 2 μg/ml 2626 (N₂)₇ luc,FAST, Cripto A10B2.18 10 μg/ml 3466 (N₂)₇ luc, FAST, Cripto A10B2.18 2μg/ml 3103

Example 4 Assay for In Vitro Inhibition of Tumor Cell Growth

Inhibition of Cripto Signaling may also be assayed by measuring thegrowth of GEO cells in soft agar. See, e.g., Ciardiello et al.,Oncogene. 1994 January; 9(1):291-8; Ciardiello et al., Cancer Res. 1991Feb. 1; 51(3):1051-4.

First, melt 3% bactoagar. Keep at 42° C. in a water bath. Then, mix 3%bactoagar solution with prewarmed complete media to make a solution of0.6% bactoagar, keeping at 42° C. Plate 4 mls of the solution in a 6 cmdish and let cool for at least 30 minutes to form the bottom agar layer.Trypsinize GEO cells and resuspend to 10⁵ cells/ml in complete media.Add antibodies to be assayed, or controls, to the cell suspensions,titrating antibodies from 20 μg to 1 μg. Mix equal volumes of the GEOcell suspensions and 0.6% bactoagar and overlay 2 mls on top of thebottom agar layer. Let cool for at least 1 hour. Incubate for 14 days at37° C. in CO₂ incubator. Count colonies visible without the use of amicroscope. The absence of colonies, as compared to negative controls,indicates that the antibody tested inhibits in vitro tumor cell growth.

This assay was used to yield the results shown in Table 4, for theantibodies A27F6.1 and B6G7.10, both of which demonstrate the ability todecrease growth of GEO cell colonies.

TABLE 4 Results of growth in soft agar assay Average number Antibody ofcolonies none 109.0 none 104.3 A27.F6 20 μg/ml 82.0 A27F6.1 10 μg/ml78.3 A27F6.1 5 μg/ml 79.0 A27F6.1 1 μg/ml 108.7 B6G7.10 20 μg ml 102.3B6G7.10 10 μg/ml 71.7

Example 5 Assay for In Vivo Inhibition of Tumor Cell Growth

To assess the inhibition of tumor cell growth, a human tumor cell lineis implanted subcutaneously in athymic nude mice and the effects of theantibodies of the invention are observed, with and without additionalchemotherapeutic treatments which may provide synergistic or additiveeffects on tumor inhibition.

This assay may be performed alternatively using different tumor celllines, such as, for example, GEO (a well differentiated human coloncancer in-vitro cell line, is obtained from the American Tissue TypeCollection (ATCC)), DU-4475 (a breast cancer in-vitro cell line obtainedfrom the ATCC), NCCIT (a testicular tumor cell line obtained from ATCC),or others known in the art. One example of such assays is as follows:

Animals are individually marked by ear punches. The GEO cell line ispassed in-vitro or in-vivo for 1-4 passages. Animals are implanted withGEO cells subcutaneously in the right flank area. The following groupsof animals may be used:

Group # Treatment # of Mice 1. Saline Control, 0.2 ml/mouse, i.p. 20three times weekly (M, W, F) 2. mAb, low dose, i.p. 10 3. mAb, middledose, i.p. 10 4. mAb, high dose, i.p. 10 5. 5-FU, 30 mg/kg/inj, i.p., 3Rx/wk 10 (M, W, F) 6. Cisplatin, 2 mg/kg/inj, s.c., 3 10 Rx/wk (M, W, F)7. Adriamycin, 1.6 mg/kg/inj, i.p., 10 3 Rx/wk (M, W, F) 8. Irinotecan,10 mg/kg/inj., i.p., 10 5 Rx/wk (M-F) 9. mAb, low dose, i.p. + 10 5-FU(intermediate dose) 10. mAb, middle dose, i.p. + 10 5-FU (intermediatedose) 11. mAb, high dose, i.p. + 10 5-FU (intermediate dose) 12. mAb,low dose, i.p. + 10 Cisplatin (intermediate dose) 13. mAb, middle dose,i.p. + 10 Cisplatin (intermediate dose) 14. mAb, high dose, i.p. + 10Cisplatin (intermediate dose) 15. mAb, low dose, i.p. + 10 Adriamycin(intermediate dose) 16. mAb, middle dose, i.p. + 10 Adriamycin(intermediate dose) 17. mAb, high dose, i.p. + 10 Adriamycin(intermediate dose) 18. mAb, low dose, i.p. + 10 Irinotecan(intermediate dose) 19. mAb, middle dose, i.p. + 10 Irinotecan(intermediate dose) 20. mAb, high dose, i.p. + 10 Irinotecan(intermediate dose)

Day 0: Implant tumor, record initial body weight of animals.

Day 1: Initiate treatments as indicated above.

Day 5: Begin tumor size and body weight measurements and continue twotimes weekly until termination of experiment.

Initial body weight, tumor size and body weight measurements, histologyat sacrifice, and immunohistochemistry analysis on tumors are examined,analyzing for Cripto expression, tumor growth, and inhibition thereof.

Example 6 In Vivo Xenograft Tumor Model—Cys-Rich Blocking Anti-CriptoAntibody

To assess the response of an NCCIT, a human testicular carcinoma cellline was implanted subcutaneously with an antibody which binds to acys-rich domain of Cripto. The experimental methods are listed below.The results are shown in FIG. 1.

Methods and Materials Animals: Athymic nude male mice were used. Animalswere individually numbered by ear punches. Tumor: NCCIT, mediastinalmixed germ cell human testicular carcinoma in-vitro cell line originallyobtained from the American Tissue Type Collection. Cell line was passedin-vitro for six passages in RPMI- 1640/10% FBS without antibiotics.Animals implanted subcutaneously with 5 × 10⁶ cells/0.2 ml matrigel onthe animals right flank. Group # Treatment # of Mice 1 Vehicle Control,(25 mM sodium 20 phosphate, 100 mM sodium chloride, pH 7.2), 0.2ml/mouse, i.p., Q14D Treatments begin on day −1 2. A8G3.5, 1 mg/kg/inj,i.p., Q14D 10 Treatments begin on day −1 3. A8G3.5, 3 mg/kg/inj, i.p.,Q14D 10 Treatments begin on day −1 4. A8G3.5, 10 mg/kg/inj, i.p., Q14D10 Treatments begin on day −1 5. Cis-platinum, 2 mg/kg/inj, s.c., 103×/wk (M, W, F) for 6 treatments Treatments began on day 1 Testingschedule Day −1: Randomized mice into control and treatments groups.Recorded initial body weight of animals. Administered first treatmentsto antibody groups. Dosing solutions were made. Treatments were blindedto the technicians until the assay was terminated. Day 0: Implantedtumor. Ran bacterial cultures on the tumor implanted into mice. Day 1:Administered first treatment to the positive chemotherapeutic group. Day4: Recorded initial tumor size measurements for tumor baseline onmatrigel. Continued to record tumor size and body weights on mice 2×/week. Monitored the study daily and made notations of any unusualobservation on animals. Endpoints: Initial body weight Tumor size andbody weight measurements

Example 7 In Vivo Xenograft Tumor Model—EGF-Like Domain BlockingAnti-Cripto Antibody

To assess the response of an NCCIT, a human testicular carcinoma cellline was implanted subcutaneously with an antibody which binds to aEGF-like domain of Cripto. The experimental methods are listed below.The results are shown in FIG. 2.

Methods and Materials: Animals: Athymic nude male mice were used.Animals were individually numbered by ear punches. Tumor: NCCIT,mediastinal mixed germ cell human testicular carcinoma in-vitro cellline originally obtained from the American Tissue Type Collection. Cellline was passed in- vitro for eight passages in RPMI-1640/10% FBSwithout antibiotics. Animals implanted subcutaneously with 5 × 10⁶cells/0.2 ml matrigel on the animals right flank. Group # Treatment # ofMice 1. Vehicle Control, (25 mM sodium 18 phosphate, 100 mM sodiumchloride, pH 7.2), 0.2 ml/mouse, i.p., Q14D Treatments begin on day −12. A27F6.1, 1 mg/kg/inj, i.p., Q14D 10 Treatments begin on day −1 with aloading dose of 2.6 mg/kg/mouse 3. A27F6.1, 10 mg/kg/inj, i.p., Q14D 10Treatments begin on day −1 with a loading dose of 21.2 mg/kg/mouse 4.Cis-platinum, 2 mg/kg/inj, s.c., 10 3×/wk (M, W, F) for 6 treatmentsTreatments began on day 1 Testing schedule Day −1: Randomized mice intocontrol and treatments groups. Recorded initial body weight of animals.Administered first treatments to antibody groups. Dosing solutions weremade. Treatments were blinded to the technicians until the assay wasterminated. Day 0: Implant tumor. Ran bacterial cultures on the tumorsimplanted into mice. Bacterial culture were negative for contaminationat 24 and 48 hours post sampling. Day 1: Administered first treatment tothe positive chemotherapeutic group. Day 4: Recorded initial tumor sizemeasurements for tumor baseline on matrigel. Continued to record tumorsize and body weights on mice 2×/week. Monitored the study daily andmade notations of any unusual observation on animals. Endpoints: Initialbody weight Tumor size and body weight measurements

Example 8 Cripto Mabs that Block ALK4 Binding

In order to assess whether Cripto-specific monoclonal antibodies caninterfere with Cripto's ability to bind to Alk4, the activin type Ireceptor, we used flow cytometry analysis using a 293 cell line whichstably expresses Alk4. To generate this cell line, 293 cells werecotransfected with a plasmid that expresses Alk4 tagged at theC-terminus with a HA epitope and a plasmid that expresses the drug,puromycin, at a 10:1 ratio. The transfected cells were then selected inpuromycin until colonies formed. Colonies were then picked, expanded andthen analyzed for Alk4 expression using western blotting analysis forHA. Clone 21 (293-Alk4-21) was found to express high levels of Alk4compared to control, untransfected 293 cells.

To analyze Cripto-Alk4 binding by flow cytometry, a purified, solubleform of human Cripto (aa 1-169) fused to the Fc portion of human IgG(CrdelC-Fc) was employed. Approximately 5 μg/ml of CrdelC-Fc or controlFc protein was incubated with 3×10⁵ 293-Alk4-21 cells on ice for 30minutes in 50 μl total volume of FACS buffer (PBS with 0.1% BSA). Forsamples containing anti-Cripto antibodies, 5 μg/ml CrdelC-Fc waspreincubated with 50 μg/ml of each Cripto antibody (A10.B2.18,A40.G12.8, A27.F6.1, A8.H3.1, A19.A10.30, A6.F8.6, A8.G3.5, A6.C12.11)on ice prior to addition of the cells. The cells were then washed inFACS buffer and the bound Fc protein was detected by incubating thecells with a R-phycoerytherin-conjugated goat anti-human IgG (Fcfragment specific) from Jackson Immunologics. Samples were then washedagain, fixed in 1% paraformaldehyde in PBS, and analyzed using standardflow cytometry procedures. The results of the FACS assay are shown inFIG. 3.

Some of the embodiments of the invention described above are outlinedbelow and include, but are not limited to, the following embodiments. Asthose skilled in the art will appreciate, numerous changes andmodifications may be made to the various embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention.

The entire disclosure of each publication cited herein is herebyincorporated by reference.

1. A method of inhibiting proliferation of tumor cells in a subject comprising the step of administering to the subject an effective amount of a composition comprising a monoclonal antibody that binds to Cripto and a pharmaceutically acceptable carrier.
 2. The method according to claim 1, wherein the subject is human.
 3. The method according to claim 1, wherein the tumor cell is selected from the group consisting of breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumor cells.
 4. The method of claim 1, wherein the antibody is a humanized antibody.
 5. The method of claim 1, wherein the antibody is a human antibody.
 6. The method of claim 1, wherein the antibody is an antibody fragment selected from the group consisting of a Fab, a Fab′, and a F(ab′)2 fragment.
 7. The method of claim 1, wherein the antibody is a full length antibody.
 8. The method of claim 1, wherein the antibody is a single chain antibody.
 9. The method of claim 1, wherein the antibody is administered in combination with a chemotherapeutic agent which is not conjugated to the antibody.
 10. The method of claim 1, wherein the antibody is conjugated to a chemotherapeutic agent.
 11. The method of claim 10, wherein the chemotherapeutic agent is selected from the group consisting of a tumor-activated prodrug, a radionuclide and a toxin.
 12. The antibody of claim 11, wherein the agent is a maytansinoid.
 13. The method of claim 1, wherein the antibody binds to an epitope comprised in the extracellular domain spanning amino acid residues 31-188 of SEQ ID NO:1 or SEQ ID NO:2.
 14. The method of claim 1, wherein the antibody binds to an epitope comprised in the ligand-receptor binding domain spanning amino acid residues 75-150 of SEQ ID NO:1 or SEQ ID NO:2.
 15. The method of claim 1, wherein the antibody binds to an epitope comprised in the EGF-like domain spanning amino acid residues 75-112 of SEQ ID NO:1 or SEQ ID NO:2.
 16. The method of claim 1, wherein the antibody binds specifically to an epitope of Cripto selected from the group of epitopes to which antibodies produced by hybridomas A7H1.19, A8F1.30, A2D3.23, A7A10.29, A9G9.9, A15C12.10, A15E4.14, A17C12.28, A17G12.1, A17H6.1, A18B3.11, and B11H8.4 bind in an effective amount.
 17. A method of treating a subject having a tumor that over-expresses Cripto comprising administering to the subject a composition comprising a monoclonal antibody that binds to Cripto and a pharmaceutically acceptable carrier in an effective amount.
 18. The method according to claim 17, wherein the subject is human.
 19. The method according to claim 17, wherein the tumor cell is selected from the group consisting of breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumor cells.
 20. The method of claim 17, wherein the antibody is a humanized antibody.
 21. The method of claim 17, wherein the antibody is a human antibody.
 22. The method of claim 17, wherein the antibody is an antibody fragment selected from the group consisting of a Fab, a Fab′, and a F(ab′)2 fragment.
 23. The method of claim 17, wherein the antibody is a full length antibody.
 24. The method of claim 17, wherein the antibody is a single chain antibody.
 25. The method of claim 17, wherein the antibody is administered in combination with a chemotherapeutic agent which is not conjugated to the antibody.
 26. The method of claim 17, wherein the antibody is conjugated to a chemotherapeutic agent.
 27. The method of claim 26, wherein the chemotherapeutic agent is selected from the group consisting of a tumor-activated prodrug, a radionuclide and a toxin.
 28. The antibody of claim 27, wherein the agent is a maytansinoid.
 29. The method of claim 17, wherein the antibody binds to an epitope comprised in the extracellular domain spanning amino acid residues 31-188 of SEQ ID NO:1 or SEQ ID NO:2.
 30. The method of claim 17, wherein the antibody binds to an epitope comprised in the ligand-receptor binding domain spanning amino acid residues 75-150 of SEQ ID NO:1 or SEQ ID NO:2.
 31. The method of claim 17, wherein the antibody binds to an epitope comprised in the EGF-like domain spanning amino acid residues 75-112 of SEQ ID NO: 1 or SEQ ID NO:2.
 32. The method of claim 17, wherein the antibody binds specifically to an epitope of Cripto selected from the group of epitopes to which antibodies produced by hybridomas A7H1.19, A8F1.30, A2D3.23, A7A10.29, A9G9.9, A15C12.10, A15E4.14, A17C12.28, A17G12.1, A17H6.1, A18B3.11 and B11H8.4 bind in an effective amount. 